Abstract:

Compounds of formula (I), or salts thereof, which inhibit acetyl
CoA(acetyl coenzyme A):diacylglycerol acyltransferase (DGAT1) activity
are provided,
##STR00001##
wherein, for example, R1 is optionally substituted phenyl or
naphthyl; Ring A is selected from (3-6C)cycloalkyl, (5-12C)bicycloalkyl
and phenyl; n is 0, 1 or 2; R2 is, for example, hydrogen, fluoro,
chloro or hydroxy; Ring B is selected from (3-6C)cycloalkyl,
(5-12C)bicycloalkyl and phenyl; L1 is a direct bond or a defined
linker group andR3 is, for example, hydroxy, carboxy or
(1-6C)alkoxycarbonyl; together with processes for their preparation,
pharmaceutical compositions containing them and their use as medicaments.

Claims:

1. A compound of formula (I) ##STR00023## or a salt thereof,
wherein:R1 is selected from phenyl and naphthyl;wherein R1 is
optionally substituted with either:i) a substituent selected from group
a) and optionally a substituent selected from group b) or up to 2
substituents from group c); orii) 1 or 2 substituents independently
selected from group b) and optionally a substituent selected from group
c); oriii) up to 3 substituents independently selected from group
c);wherein groups a) to c) are as follows:group a) (2-4C)alkyl,
(2-4C)alkenyl, (2-4C)alkynyl, phenyl, 4-fluorophenyl, phenoxy,
4-fluorophenoxy, benzyloxy, 4-fluorobenzyloxy, (3-6C)cycloalkyl,
(3-6C)cycloalkoxy, halo(1-2C)alkyl;group b) chloro, methyl and
methoxy;group c) fluoro;Ring A is selected from (3-6C)cycloalkyl,
(5-12C)bicycloalkyl and phenyl;n is 0, 1 or 2;R2 is selected from
hydrogen, fluoro, chloro, hydroxy, methoxy, halo(1-2C)alkyl, methyl,
ethyl, cyano and methylsulfonyl;Ring B is selected from (3-6C)cycloalkyl,
(5-12C)bicycloalkyl and phenyl;L1 is a direct bond or a linker
selected from --(CR4R5)1-2--,
--O--(CR4R5)1-2-- and
--CH2(CR4R5)1-2--, wherein for each value of L1,
the CR4R5 group is directly attached to R3;each R4 is
independently selected from hydrogen, hydroxy, (1-3C)alkoxy, (1-4C)alkyl,
hydroxy(1-3C)alkyl and (1-2C)alkoxy(1-2C)alkyl; provided that when
L2 is --O--(CR4R5)1-2--then the R4 on the carbon
atom directly attached to the oxygen atom is not hydroxy or
(1-3C)alkoxy;each R5 is independently selected from hydrogen and
methyl;R3 is selected from hydroxy, carboxy, (1-6C)alkoxycarbonyl,
SO3H, --S(O)2NHR13, --S(O)2NHC(O)R13,
--CH2S(O)2R13, --C(O)NHS(O)2R13, --C(O)NHOH,
--C(O)NHCN, --CH(CF3)OH, C(CF3)2OH, --P(O)(OH), and a
5-membered heterocyclic ring selected from the group consisting of
##STR00024## ##STR00025## R13 is (1-6C)alkyl, aryl or heteroaryl;
andR27 is hydrogen or (1-4C)alkyl.

2. The compound according to claim 1 which is selected from
4-[4-[[5-[(3,4,5-trifluorophenyl)amino]1,3,4-oxadiazole-2-carbonyl]amino]-
phenyl]sulfonylcyclohexane-1-carboxylic acid;
4-[4-[[5-[(4-fluorophenyl)amino]1,3,4-oxadiazole-2-carbonyl]amino]phenyl]-
sulfonylcyclohexane-1-carboxylic acid and a pharmaceutically-acceptable
salt of either of these.

3. (canceled)

4. A method for producing an inhibition of DGAT1 activity in a
warm-blooded animal in need of such treatment, comprising administering
to the animal an effective amount of a compound of formula (I) as claimed
in claim 1 or a pharmaceutically-acceptable salt thereof.

5. A method of treating diabetes mellitus and/or obesity in a warm-blooded
animal in need of such treatment, comprising administering to the animal
an effective amount of a compound of formula (I) as claimed in claim 1 or
a pharmaceutically-acceptable salt thereof.

6-7. (canceled)

8. A pharmaceutical composition comprising a compound of formula (I) as
claimed in claim 1 or a pharmaceutically-acceptable salt thereof, in
association with a pharmaceutically-acceptable excipient or carrier.

9. A process for preparing a compound according to claim 1 comprising one
of the following steps, wherein all variables are as hereinbefore defined
for a compound of formula (I) unless otherwise stated:a) reacting a
compound of formula (I) to form another compound of formula (I);b)
cyclising a compound of formula (2) ##STR00026## c) reacting an amine of
formula (3) with a carboxylate of formula (4): ##STR00027## and
optionally thereafter:i) removing any protecting groups; and/orii)
forming a salt thereof.

Description:

[0001]The present invention relates to compounds which inhibit acetyl CoA
(acetyl coenzyme A):diacylglycerol acyltransferase (DGAT1) activity,
processes for their preparation, pharmaceutical compositions containing
them as the active ingredient, methods for the treatment of disease
states associated with DGAT1 activity, to their use as medicaments and to
their use in the manufacture of medicaments for use in the inhibition of
DGAT1 in warm-blooded animals such as humans. In particular this
invention relates to compounds useful for the treatment of type H
diabetes, insulin resistance, impaired glucose tolerance and obesity in
warm-blooded animals such as humans, more particularly to the use of
these compounds in the manufacture of medicaments for use in the
treatment of type II diabetes, insulin resistance, impaired glucose
tolerance and obesity in warm-blooded animals such as humans.

[0002]Acyl CoA:diacylglycerol acyltransferase (DGAT) is found in the
microsomal fraction of cells. It catalyzes the final reaction in the
glycerol phosphate pathway, considered to be the main pathway of
triglyceride synthesis in cells by facilitating the joining of a
diacylglycerol with a fatty acyl CoA, resulting in the formation of
triglyceride. Although it is unclear whether DGAT is rate-limiting for
triglyceride synthesis, it catalyzes the only step in the pathway that is
committed to producing this type of molecule [Lehner & Kuskis (1996)
Biosynthesis of triacylglycerols. Prog. Lipid Res. 35: 169-201].

[0003]Two DGAT genes have been cloned and characterised. Both of the
encoded proteins catalyse the same reaction although they share no
sequence homology. The DGAT1 gene was identified from sequence database
searches because of its similarity to acyl CoA:cholesterol
acyltransferase (ACAT) genes. [Cases et al (1998) Identification of a
gene encoding an acyl CoA:diacylglycerol acyltransferase, a key enzyme in
triacylglycerol synthesis. Proc. Natl. Acad. Sci. USA 95: 13018-13023].
DGAT1 activity has been found in many mammalian tissues, including
adipocytes.

[0004]Because of the previous lack of molecular probes, little is known
about the regulation of DGAT1. DGAT1 is known to be significantly
up-regulated during adipocyte differentiation.

[0005]Studies in gene knockout mice has indicated that modulators of the
activity of DGAT1 would be of value in the treatment of type II diabetes
and obesity. DGAT1 knockout (Dgat1.sup.-/-) mice, are viable and capable
of synthesizing triglycerides, as evidenced by normal fasting serum
triglyceride levels and normal adipose tissue composition. Dgat1.sup.-/-
mice have less adipose tissue than wild-type mice at baseline and are
resistant to diet-induced obesity. Metabolic rate is ˜20% higher in
Dgat1.sup.-/- mice than in wild-type mice on both regular and high-fat
diets [Smith et al (2000) Obesity resistance and multiple mechanisms of
triglyceride synthesis in mice lacking DGAT. Nature Genetics 25: 87-90].
Increased physical activity in Dgat1.sup.-/- mice partially accounts for
their increased energy expenditure. The Dgat1.sup.-/- mice also exhibit
increased insulin sensitivity and a 20% increase in glucose disposal
rate. Leptin levels are 50% decreased in the Dgat1.sup.-/- mice in line
with the 50% decrease in fat mass.

[0009]Accordingly, the present invention provides a compound of formula
(I)

##STR00002##

or a salt thereof, wherein:R1 is selected from phenyl and
naphthyl;wherein R1 is optionally substituted with either:i) a
substituent selected from group a) and optionally a substituent selected
from group b) or up to 2 substituents from group c); orii) 1 or 2
substituents independently selected from group b) and optionally a
substituent selected from group c); oriii) up to 3 substituents
independently selected from group c);wherein groups a) to c) are as
follows:group a) (2-4C)alkyl, (2-4C)alkenyl, (2-4C)alkynyl, phenyl,
4-fluorophenyl, phenoxy, 4-fluorophenoxy, benzyloxy, 4-fluorobenzyloxy,
(3-6C)cycloalkyl, (3-6C)cycloalkoxy, halo(1-2C)alkyl;group b) chloro,
methyl and methoxy;group c) fluoro;Ring A is selected from
(3-6C)cycloalkyl, (5-12C)bicycloalkyl and phenyl;n is 0, 1 or 2;R2
is selected from hydrogen, fluoro, chloro, hydroxy, methoxy,
halo(1-2C)alkyl, methyl, ethyl, cyano and methylsulfonyl;Ring B is
selected from (3-6C)cycloalkyl, (5-12C)bicycloalkyl and phenyl;L1 is
a direct bond or is a linker selected from --(CR4R5)1-2--,
--O--(CR4R5)1-2-- and
--CH2(CR4R5)1-2-- (wherein for each value of L1,
the CR4R5 group is directly attached to R3); each R4
is independently selected from hydrogen, hydroxy, (1-3C)alkoxy,
(1-4C)alkyl, hydroxy(1-3C)alkyl and (1-2C)alkoxy(1-2C)alkyl; provided
that when L2 is --O--(CR4R5)1-2-- then the R4 on
the carbon atom directly attached to the oxygen atom is not hydroxy or
(1-3C)alkoxy;each R5 is independently selected from hydrogen and
methyl;R3 is selected from hydroxy, carboxy, (1-6C)alkoxycarbonyl,
and a carboxylic acid mimic or bioisostere.

[0010]In this specification the term "alkyl" includes both straight and
branched chain alkyl groups but references to individual alkyl groups
such as "propyl" are specific for the straight chain version only. An
analogous convention applies to other generic terms. Unless otherwise
stated the term "alkyl" advantageously refers to chains with 1-10 carbon
atoms, suitably from 1-6 carbon atoms, preferably 1-4 carbon atoms.

[0011]In this specification the term "alkoxy" means an alkyl group as
defined hereinbefore linked to an oxygen atom.

[0012]It is to be understood that optional substituents on any group may
be attached to any available atom as appropriate unless otherwise
specified, including heteroatoms provided that they are not thereby
quaternised.

[0013]In this specification the term "heteroatom" refers to non-carbon
atoms such as oxygen, nitrogen or sulphur atoms.

[0014]Unless specified otherwise, the expression "haloalkyl" refers to
alkyl groups which carry at least one halo substitutent. This includes
perhalo groups where all hydrogen atoms are replaced by halo such as
fluoro.

[0015]It is to be understood that optional substituents on any group may
be attached to any available atom as appropriate unless otherwise
specified, including heteroatoms provided that they are not thereby
quaternised.

[0016]Within this specification composite terms are used to describe
groups comprising more than one functionality such as
(1-6C)alkoxy(1-6C)alkyl. Such terms are to be interpreted in accordance
with the meaning which is understood by a person skilled in the art for
each component part.

[0017]Where optional substituents are chosen from "0, 1, 2 or 3" groups it
is to be understood that this definition includes all substituents being
chosen from one of the specified groups or the substituents being chosen
from two or more of the specified groups. An analogous convention applies
to substituents chosen from "0, 1 or 2" groups and "1 or 2" and any other
analogous groups.

[0018]Substituents may be present at any suitable position on, for
example, an alkyl group. Therefore, hydroxy substituted (1-6C)alkyl
includes hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl and
3-hydroxypropyl.

[0019]Examples of (1-4C)alkyl include methyl, ethyl, propyl and isopropyl;
examples of (1-6C)alkyl include methyl, ethyl, propyl, isopropyl,
t-butyl, pentyl, iso-pentyl, 1-2-dimethylpropyl and hexyl; examples of
(2-4C)alkyl include ethyl, propyl, isopropyl, t-butyl; examples of
(2-4C)alkenyl include ethenyl, propenyl, but-2-enyl and but-1-enyl;
examples of (2-4C)alkynyl include ethynyl, propynyl, but-2-ynyl and
but-1-ynyl; examples of (1-3C)alkoxy include methoxy, ethoxy, propoxy and
isopropoxy; examples of (1-4C)alkoxy include methoxy, ethoxy, propoxy,
isopropoxy and tert-butoxy; examples of (1-6C)alkoxy include methoxy,
ethoxy, propoxy, isopropoxy, tert-butoxy and pentoxy; examples of
(1-2C)alkoxy(1-2C)alkyl include methoxymethyl, ethoxymethyl and
methoxyethyl; examples of (3-6C)cycloalkyl cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl; examples of (3-6C)cycloalkyloxy cyclopropyloxy,
cyclobutyloxy, cyclopentyloxy and cyclohexyloxy; examples of
(5-12C)bicycloalkyl include norbornyl, decalinyl (bicyclo[4,4,0]decyl
(cis and trans), bicyclo[5,3,0]decyl and hydrindanyl
(bicyclo[4,3,0]nonyl); examples of halo are chloro, bromo, iodo and
fluoro; examples of halo(1-6C)alkyl include halo(1-4C)alkyl such as
chloromethyl, fluoroethyl, fluoromethyl, fluoropropyl; fluorobutyl,
dichloromethyl, difluoromethyl, 1,2-difluoroethyl and 1,1-difluoroethyl
as well as perhalo(1-6C)alkyl (including perhalo(1-4C)alkyl) such as
trifluoromethyl, pentafluoroethyl, and heptafluoropropyl; examples of
halo(1-2C)alkyl include fluoromethyl, difluoromethyl, trifluoromethyl,
fluoroethyl and pentafluoroethyl; examples of hydroxy(1-6C)alkyl include
hydroxy(1-3C)alkyl such as hydroxy methyl, 1-hydroxyethyl, 2-hydroxyethyl
and 3-hydroxypropyl; examples of (1-6C)alkoxycarbonyl
(N-(1-6C)alkylcarbamoyl) include (1-4C)alkcoxycarbonyl such as
methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, iso-propoxycarbonyl and
tert-butoxycarbonyl;

wherein R13 is (1-6C)alkyl, aryl or heteroaryl; and R27 is
hydrogen or (1-4C)alkyl. It will be understood that in the above
sub-formulae (a) to (i'), keto-enol tautomerism may be possible and that
the sub-formulae (a) to (i') should be taken to encompass all tautomers
thereof.

[0021]For the avoidance of doubt it is to be understood that where in this
specification a group is qualified by `hereinbefore defined` or `defined
hereinbefore` the said group encompasses the first occurring and broadest
definition as well as each and all of the particular definitions for that
group.

[0022]It is to be understood that where substituents contain two
substituents on an alkyl chain, in which both are linked by a heteroatom
(for example two alkoxy substituents), then these two substituents are
not substituents on the same carbon atom of the alkyl chain.

[0023]If not stated elsewhere, suitable optional substituents for a
particular group are those as stated for similar groups herein.

[0024]A compound of formula (I) may form stable acid or basic salts, and
in such cases administration of a compound as a salt may be appropriate,
and pharmaceutically acceptable salts may be made by conventional methods
such as those described following.

[0025]Suitable pharmaceutically-acceptable salts include acid addition
salts such as methanesulfonate, tosylate, α-glycerophosphate,
fumarate, hydrochloride, citrate, maleate, tartrate and (less preferably)
hydrobromide. Also suitable are salts formed with phosphoric and sulfuric
acid. In another aspect suitable salts are base salts such as a group (I)
(alkali metal) salt, a group (II) (alkaline earth metal) salt, an organic
amine salt for example triethylamine, morpholine, N-methylpiperidine,
N-ethylpiperidine, procaine, dibenzylamine, N,N-dibenzylethylamine,
tris-(2-hydroxyethyl)amine, N-methyl d-glucamine and amino acids such as
lysine. There may be more than one cation or anion depending on the
number of charged functions and the valency of the cations or anions.

[0026]However, to facilitate isolation of the salt during preparation,
salts which are less soluble in the chosen solvent may be preferred
whether pharmaceutically-acceptable or not.

[0027]Within the present invention it is to be understood that a compound
of the formula (I) or a salt thereof may exhibit the phenomenon of
tautomerism and that the formulae drawings within this specification can
represent only one of the possible tautomeric forms. It is to be
understood that the invention encompasses any tautomeric form which
inhibits DGAT1 activity and is not to be limited merely to any one
tautomeric form utilised within the formulae drawings.

[0028]Pro-drugs of compounds of formula (I) are also within the scope of
the invention.

[0029]Various forms of prodrugs are known in the art. For examples of such
prodrug derivatives, see:

[0030]Examples of such prodrugs are in vivo cleavable esters of a compound
of the invention. An in vivo cleavable ester of a compound of the
invention containing a carboxy group is, for example, a
pharmaceutically-acceptable ester which is cleaved in the human or animal
body to produce the parent acid. Suitable pharmaceutically-acceptable
esters for carboxy include (1-6C)alkyl esters, for example methyl or
ethyl; (1-6C)alkoxymethyl esters, for example methoxymethyl;
(1-6C)alkanoyloxymethyl esters, for example pivaloyloxymethyl; phthalidyl
esters; (3-8C)cycloalkoxycarbonyloxy(1-6C)alkyl esters, for example
1-cyclohexylcarbonyloxyethyl; 1,3-dioxolan-2-ylmethyl esters, for example
5-methyl-1,3-dioxolan-2-ylmethyl; (1-6C)alkoxycarbonyloxyethyl esters,
for example 1-methoxycarbonyloxyethyl; aminocarbonylmethyl esters and
mono- or di- N-((1-6C)alkyl) versions thereof, for example
N,N-dimethylaminocarbonylmethyl esters and N-ethylaminocarbonylmethyl
esters; and may be formed at any carboxy group in the compounds of this
invention. An in vivo cleavable ester of a compound of the invention
containing a hydroxy group is, for example, a pharmaceutically-acceptable
ester which is cleaved in the human or animal body to produce the parent
hydroxy group. Suitable pharmaceutically acceptable esters for hydroxy
include (1-6C)alkanoyl esters, for example acetyl esters; and benzoyl
esters wherein the phenyl group may be substituted with aminomethyl or
N-substituted mono- or di-(1-6C)allyl aminomethyl, for example
4-aminomethylbenzoyl esters and 4-N,N-dimethylaminomethylbenzoyl esters.

[0031]It will be appreciated by those skilled in the art that certain
compounds of formula (I) contain asymmetrically substituted carbon and/or
sulfur atoms, and accordingly may exist in, and be isolated in,
optically-active and racemic forms. Some compounds may exhibit
polymorphism. It is to be understood that the present invention
encompasses any racemic, optically-active, polymorphic or stereoisomeric
form, or mixtures thereof, which form possesses properties useful in the
inhibition of DGAT1 activity, it being well known in the art how to
prepare optically-active forms (for example, by resolution of the racemic
form by recrystallization techniques, by synthesis from optically-active
starting materials, by chiral synthesis, by enzymatic resolution, by
biotransformation, or by chromatographic separation using a chiral
stationary phase) and how to determine efficacy for the inhibition of
DGAT1 activity by the standard tests described hereinafter.

[0032]It is also to be understood that certain compounds of the formula
(I) and salts thereof can exist in solvated as well as =solvated forms
such as, for example, hydrated forms. It is to be understood that the
invention encompasses all such solvated forms which inhibit DGAT1
activity.

[0033]As stated before, we have discovered a range of compounds that have
good DGAT1 inhibitory activity. They have good physical and/or
pharmacokinetic properties in general.

[0034]Particular aspects of the invention comprise a compound of formula
(I), or a salt thereof, wherein the substituents R1 to R5 and
other substituents mentioned above have values defined hereinbefore, or
any of the following values (which may be used where appropriate with any
of the definitions and embodiments disclosed hereinbefore or
hereinafter):

[0035]In one embodiment of the invention there are provided compounds of
formula (I), in an alternative embodiment there are provided salts,
particularly pharmaceutically-acceptable salts, of compounds of formula
(I). In a further embodiment, there are provided pro-drugs, particularly
in-vivo cleavable esters, of compounds of formula (I). In a further
embodiment, there are provided salts, particularly
pharmaceutically-acceptable salts of pro-drugs of compounds of formula
(I).

[0036]Particular values of variable groups in compounds of formulae (I)
are as follows. Such values may be used where appropriate with any of the
other values, definitions, aspects, claims or embodiments defined
hereinbefore or hereinafter.

1) R1 is phenyl2) R1 is naphthyl3) R1 is substituted with 1
substituent4) R1 is substituted with 1, 2 or 3 fluoro5) R1 is
substituted with a substituent selected from phenyl, 4-fluorophenyl,
phenoxy, 4-fluorophenoxy, benzyloxy and 4-fluorobenzyloxy and optionally
also substituted by 1 or 2 fluoro6) R1 is substituted with a
substituent selected from (3-6C)cycloalkyl and (3-6C)cycloalkoxy and
optionally also substituted by 1 or 2 fluoro7) R1 is substituted
with a substituent selected from (2-4C)alkyl, (2-4C)alkenyl,
(2-4C)alkynyl and optionally also substituted by 1 or 2 fluoro8) R1
is substituted with a substituent selected from halo(1-2C)alkyl and
optionally also substituted by 1 or 2 fluoro9) R1 is substituted
with a substituent selected from chloro, methyl and methoxy and
optionally also substituted by 1 or 2 fluoro10) Ring A is phenyl11) Ring
A is cycloalkyl, for example cyclohexyl12) Ring B is phenyl12) Ring B is
cycloalkyl, for example cyclobutyl, cyclopentyl or cyclohexyl14) Ring B
is cyclohexyl15) Ring A is phenyl and Ring B is cyclohexyl16) L1 is
a direct bond

17) L1 is --(CR4R5)1-2--

18) L1 is --O--(CR4R5)1-2--

19) L1 is --CH2(CR4R5)1-2--

20) CR4R5 is CH2

21) CR4R5 is CHMe

22) CR4R5 is CMe2

23) CR4R5 is CH2CH(OH)

24) CR4R5 is CH2CH(CH2OH)

25) CR4R5 is CH2CH(CH2OMe)

26) CR4R5 is CH(OH)

27) CR4R5 is CH(OMe)

[0037]28) L1 is a direct bond or --CH2--29) R3 is
hydroxy30) R3 is carboxy

31) R3 is (1-6C)alkoxycarbonyl

[0038]32) R3 is a carboxylic acid mimic or bioisostere33) R3 is
carboxy or (1-6C)alkoxycarbonyl34) R2 is hydrogen or fluoro35)
R2 is hydrogen36) n is 037) n is 138) n is 2

[0039]In one aspect of the invention, there is provided a compound of
formula (I) or a salt thereof, wherein

R1 is phenyl substituted with 1, 2 or 3 fluoro;Ring A is
phenyl;R2 is hydrogen, fluoro or chloro, particularly hydrogen;n is
2;Ring B is (3-6C)cycloalkyl, such as cyclohexyl;L1 is a direct bond
or --CH2--;R3 is carboxy or methoxycarbonyl.

[0040]Further particular compounds of the invention are each of the
Examples, each of which provides a further independent aspect of the
invention. In further aspects, the present invention also comprises any
two or more compounds of the Examples.

[0041]Particular compounds of the invention are any one or more of the
following, or salts thereof:
[0042]4-[4-[[5-[(3,4,5-trifluorophenyl)amino]1,3,4-oxadiazole-2-carbonyl]-
amino]phenyl]sulfonylcyclohexane-1-carboxylic acid; and
[0043]4-[4-[[5-[(4-fluorophenyl)amino]1,3,4-oxadiazole-2-carbonyl]amino]p-
henyl]sulfonylcyclohexane-1-carboxylic acid.

[0044]Intermediates 1 and 2 in the Examples hereinafter are also within
the scope of the invention and are thus each provided as further
independent aspects.

Process

[0045]A compound of formula (I) and its salts may be prepared by any
process known to be applicable to the preparation of chemically related
compounds. Such processes, when used to prepare a compound of the formula
(I), or a salt thereof, are provided as a further feature of the
invention.

[0046]In a further aspect the present invention also provides that the
compounds of the formula (I) and salts thereof, can be prepared by a
process a) to c) as follows (wherein all variables are as hereinbefore
defined for a compound of formula (I) unless otherwise stated):

[0047]a) reaction of a compound of formula (I) to form another compound of
formula (I);

[0048]b) cyclisation of a compound of formula (2);

##STR00007##

[0049]c) reaction of an amine of formula (3) with a carboxylate of formula
(4):

##STR00008##

and thereafter if necessary or desirable:i) removing any protecting
groups; and/orii) forming a salt thereof.

Process a)

[0050]Examples of conversions of a compound of formula (I) into another
compound of Formula (I), well known to those skilled in the art, include
functional group interconversions such as hydrolysis (in particular ester
hydrolysis), oxidation (such as oxidation of a sulfide to a sulfoxide or
sulfone) or reduction (such as the reduction of an acid to an alcohol),
and/or further functionalisation by standard reactions such as amide or
metal-catalysed coupling, or nucleophilic displacement reactions.

Process b)

[0051]Compounds of formula (2) may be made as illustrated in Scheme 1
below.

##STR00009##

[0052]The process illustrate in Scheme 1 may be used analogously to
prepare compounds of formula (2) wherein Ring B is phenyl.

[0053]Compounds of formula (2) may be made by reaction of an aminocarbonyl
acylhydrazine (5) with an isothiocyanate R1NCS or isothiocyanate
equivalent such as aminothiocarbonylimidazole in a suitable solvent such
as DMF or MeCN at a temperature between 0 and 100° C. The
preparation of aminocarbonyl acylhydrazines from anilines is well known
in the art. For example reaction of an aniline (such as 7) with methyl
chlorooxoacetate in the presence of pyridine in a suitable solvent such
as DCM followed by reaction with hydrazine in a suitable solvent such as
ethanol at a temperature between 0 and 100° C.

[0054]Anilines such as compound 7 may be prepared by reduction of a nitro
compound such as compound 8, which itself may be made by oxidation of a
thiol ether such as compound 9. Compounds such as 9 may be prepared by
esterification of a compound 10, which may be prepared by reaction of the
appropriate mercaptocycloalkanecarboxylic acid (see for example Can. J.
Chem. 64, 2184 (1986)) with an appropriately substituted
4-fluoronitrobenzene.

[0055]Compounds of formula (2) wherein Ring A is cyclohexyl may be
prepared as illustrated in Scheme 2, in which Ring B is shown as
cyclohexyl, although the method may also be applied to Ring B as phenyl,
and in which LG is a leaving group such as p-toluene sulfonate
(tosylate), chloro, bormo or iodo;

##STR00010##

[0056]The compound of formula (2) may then be cyclised using, for example
agents such as carbonyldiimidazole, or tosyl chloride and a suitable base
(such as triethylamine), under conditions known in the art.

[0057]Isocyanates R1--NCO are commercially available or may be made
by reaction of the acid chlorides R1--NH2 with for example
phosgene or a phosgene equivalent followed by a suitable base (such as
triethylamine).

Process c)

[0058]Compounds of formula (3) (such as a compound of formula (7) or (11))
may be made as illustrated in Schemes 1 to 2 above.

[0059]Compounds of formula (4) may be made by alkaline hydrolysis of ester
(12) as prepared using a published procedure (J. Het. Chem. 1977, 14,
1385-1388). Ester (12) may be made by cyclisation of a compound of
formula (13) (where X is O or S) in a similar manner as described in
process b) for compounds of formula (2).

##STR00011##

[0060]An alternative method for making compounds of formula (12) is
illustrated below:

##STR00012##

[0061]Compounds of formula (3) may be coupled with compounds of formula
(4) under standard conditions for formation of amide bonds. For example
using an appropriate coupling reaction, such as a carbodiimide coupling
reaction performed with EDAC, optionally in the presence of DMAP, in a
suitable solvent such as DCM, chloroform or DMF at room temperature.

[0062]Compounds of formula (I) wherein n is 1 or 2 (that is, sulfoxides
and sulfones) may be made by oxidation of the equivalent compound wherein
n is 0 (sulfides) under standard conditions. This oxidation may be
carried out early in the synthetic process (as illustrated for example in
Scheme 1), or particularly in the case of process c), should be carried
out on the compound of formula (I) itself.

[0063]It will be appreciated that certain of the various ring substituents
in the compounds of the present invention, for example R2, may be
introduced by standard aromatic substitution reactions or generated by
conventional functional group modifications either prior to or
immediately following the processes mentioned above, and as such are
included in the process aspect of the invention. Such reactions may
convert one compound of the formula (I) into another compound of the
formula (I). Such reactions and modifications include, for example,
introduction of a substituent by means of an aromatic substitution
reaction, reduction of substituents, alkylation of substituents and
oxidation of substituents. The reagents and reaction conditions for such
procedures are well known in the chemical art. Particular examples of
aromatic substitution reactions include the introduction of a nitro group
using concentrated nitric acid, the introduction of an acyl group using,
for example, an acyl halide and Lewis acid (such as aluminium
trichloride) under Friedel Crafts conditions; the introduction of an
alkyl group using an alkyl halide and Lewis acid (such as aluminium
trichloride) under Friedel Crafts conditions; and the introduction of a
halogen group. Particular examples of modifications include the reduction
of a nitro group to an amino group by for example, catalytic
hydrogenation with a nickel catalyst or treatment with iron in the
presence of hydrochloric acid with heating; oxidation of alkylthio to
alkanesulfinyl or alkanesulfonyl.

[0064]If not commercially available, the necessary starting materials for
the procedures such as those described above may be made by procedures
which are selected from standard organic chemical techniques, techniques
which are analogous to the synthesis of known, structurally similar
compounds, techniques which are described or illustrated in the
references given above, or techniques which are analogous to the above
described procedure or the procedures described in the examples. The
reader is further referred to Advanced Organic Chemistry, 5th
Edition, by Jerry March and Michael Smith, published by John Wiley & Sons
2001, for general guidance on reaction conditions and reagents.

[0065]It will be appreciated that some intermediates to compounds of the
formula (I) are also novel and these are provided as separate independent
aspects of the invention. In particular, certain compounds of formulae
(2), (3), (5), (6) and/or (7) are each provided as independent aspects of
the invention.

[0066]It will also be appreciated that in some of the reactions mentioned
herein it may be necessary/desirable to protect any sensitive groups in
compounds. The instances where protection is necessary or desirable are
known to those skilled in the art, as are suitable methods for such
protection. Conventional protecting groups may be used in accordance with
standard practice (for illustration see T. W. Greene, Protective Groups
in Organic Synthesis, John Wiley and Sons, 1991).

[0067]Protecting groups may be removed by any convenient method as
described in the literature or known to the skilled chemist as
appropriate for the removal of the protecting group in question, such
methods being chosen so as to effect removal of the protecting group with
minimum disturbance of groups elsewhere in the molecule.

[0068]Thus, if reactants include, for example, groups such as amino,
carboxy or hydroxy it may be desirable to protect the group in some of
the reactions mentioned herein.

[0069]Examples of a suitable protecting group for a hydroxy group is, for
example, an acyl group, for example an alkanoyl group such as acetyl, an
aroyl group, for example benzoyl, a silyl group such as trimethylsilyl or
an arylmethyl group, for example benzyl. The deprotection conditions for
the above protecting groups will necessarily vary with the choice of
protecting group. Thus, for example, an acyl group such as an alkanoyl or
an aroyl group may be removed, for example, by hydrolysis with a suitable
base such as an alkali metal hydroxide, for example lithium or sodium
hydroxide. Alternatively a silyl group such as trimethylsilyl or SEM may
be removed, for example, by fluoride or by aqueous acid; or an arylmethyl
group such as a benzyl group may be removed, for example, by
hydrogenation in the presence of a catalyst such as palladium-on-carbon.

[0070]A suitable protecting group for an amino group is, for example, an
acyl group, for example an alkanoyl group such as acetyl, an
alkoxycarbonyl group, for example a methoxycarbonyl, ethoxycarbonyl or
tert-butoxycarbonyl group, an arylmethoxycarbonyl group, for example
benzyloxycarbonyl, or an aroyl group, for example benzoyl. The
deprotection conditions for the above protecting groups necessarily vary
with the choice of protecting group. Thus, for example, an acyl group
such as an alkanoyl or alkoxycarbonyl group or an aroyl group may be
removed for example, by hydrolysis with a suitable base such as an alkali
metal hydroxide, for example lithium or sodium hydroxide. Alternatively
an acyl group such as a t-butoxycarbonyl group may be removed, for
example, by treatment with a suitable acid as hydrochloric, sulfuric or
phosphoric acid or trifluoroacetic acid and an arylmethoxycarbonyl group
such as a benzyloxycarbonyl group may be removed, for example, by
hydrogenation over a catalyst such as palladium-on-carbon, or by
treatment with a Lewis acid for example boron tris(trifluoroacetate). A
suitable alternative protecting group for a primary amino group is, for
example, a phthaloyl group which may be removed by treatment with an
alkylamine, for example dimethylaminopropylamine or 2-hydroxyethylamine,
or with hydrazine.

[0071]A suitable protecting group for a carboxy group is, for example, an
esterifying group, for example a methyl or an ethyl group which may be
removed, for example, by hydrolysis with a base such as sodium hydroxide,
or for example a t-butyl group which may be removed, for example, by
treatment with an acid, for example an organic acid such as
trifluoroacetic acid, or for example a benzyl group which may be removed,
for example, by hydrogenation over a catalyst such as
palladium-on-carbon.

[0072]Resins may also be used as a protecting group.

[0073]The protecting groups may be removed at any convenient stage in the
synthesis using conventional techniques well known in the chemical art,
or they may be removed during a later reaction step or work-up.

[0074]The skilled organic chemist will be able to use and adapt the
information contained and referenced within the above references, and
accompanying Examples therein and also the examples herein, to obtain
necessary starting materials, and products.

[0075]The removal of any protecting groups and the formation of a
(pharmaceutically-acceptable) salt are within the skill of an ordinary
organic chemist using standard techniques. Furthermore, details on the
these steps has been provided hereinbefore.

[0076]When an optically active form of a compound of the invention is
required, it may be obtained by carrying out one of the above procedures
using an optically active starting material (formed, for example, by
asymmetric induction of a suitable reaction step), or by resolution of a
racemic form of the compound or intermediate using a standard procedure,
or by chromatographic separation of diastereoisomers (when produced)
Enzymatic techniques may also be useful for the preparation of optically
active compounds and/or intermediates.

[0077]Similarly, when a pure regioisomer of a compound of the invention is
required, it may be obtained by carrying out one of the above procedures
using a pure regioisomer as a starting material, or by resolution of a
mixture of the regioisomers or intermediates using a standard procedure.

[0078]According to a further aspect of the invention there is provided a
pharmaceutical composition which comprises a compound of formula (I) as
defined hereinbefore or a pharmaceutically-acceptable salt thereof, in
association with a pharmaceutically-acceptable excipient or carrier.

[0079]The compositions of the invention may be in a form suitable for oral
use (for example as tablets, lozenges, hard or soft capsules, aqueous or
oily suspensions, emulsions, dispersible powders or granules, syrups or
elixirs), for topical use (for example as creams, ointments, gels, or
aqueous or oily solutions or suspensions), for administration by
inhalation (for example as a finely divided powder or a liquid aerosol),
for administration by insufflation (for example as a finely divided
powder) or for parenteral administration (for example as a sterile
aqueous or oily solution for intravenous, subcutaneous, intramuscular or
intramuscular dosing or as a suppository for rectal dosing).

[0080]The compositions of the invention may be obtained by conventional
procedures using conventional pharmaceutical excipients, well known in
the art. Thus, compositions intended for oral use may contain, for
example, one or more colouring, sweetening, flavouring and/or
preservative agents.

[0081]Suitable pharmaceutically acceptable excipients for a tablet
formulation include, for example, inert diluents such as lactose, sodium
carbonate, calcium phosphate or calcium carbonate, granulating and
disintegrating agents such as corn starch or algenic acid; binding agents
such as starch; lubricating agents such as magnesium stearate, stearic
acid or talc; preservative agents such as ethyl or propyl
p-hydroxybenzoate, and anti-oxidants, such as ascorbic acid. Tablet
formulations may be uncoated or coated either to modify their
disintegration and the subsequent absorption of the active ingredient
within the gastrointestinal tract, or to improve their stability and/or
appearance, in either case, using conventional coating agents and
procedures well known in the art.

[0082]Compositions for oral use may be in the form of hard gelatin
capsules in which the active ingredient is mixed with an inert solid
diluent, for example, calcium carbonate, calcium phosphate or kaolin, or
as soft gelatin capsules in which the active ingredient is mixed with
water or an oil such as peanut oil, liquid paraffin, or olive oil.

[0083]Aqueous suspensions generally contain the active ingredient in
finely powdered form together with one or more suspending agents, such as
sodium carboxymethylcellulose, methylcellulose,
hydroxypropylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum
tragacanth and gum acacia; dispersing or wetting agents such as lecithin
or condensation products of an alkylene oxide with fatty acids (for
example polyoxethylene stearate), or condensation products of ethylene
oxide with long chain aliphatic alcohols, for example
heptadecaethyleneoxycetanol, or condensation products of ethylene oxide
with partial esters derived from fatty acids and a hexitol such as
polyoxyethylene sorbitol monooleate, or condensation products of ethylene
oxide with long chain aliphatic alcohols, for example
heptadecaethyleneoxycetanol, or condensation products of ethylene oxide
with partial esters derived from fatty acids and a hexitol such as
polyoxyethylene sorbitol monooleate, or condensation products of ethylene
oxide with partial esters derived from fatty acids and hexitol
anhydrides, for example polyethylene sorbitan monooleate. The aqueous
suspensions may also contain one or more preservatives (such as ethyl or
propyl p-hydroxybenzoate, anti-oxidants (such as ascorbic acid),
colouring agents, flavouring agents, and/or sweetening agents (such as
sucrose, saccharine or aspartame).

[0084]Oily suspensions may be formulated by suspending the active
ingredient in a vegetable oil (such as arachis oil, olive oil, sesame oil
or coconut oil) or in a mineral oil (such as liquid paraffin). The oily
suspensions may also contain a thickening agent such as beeswax, hard
paraffin or cetyl alcohol. Sweetening agents such as those set out above,
and flavouring agents may be added to provide a palatable oral
preparation. These compositions may be preserved by the addition of an
anti-oxidant such as ascorbic acid.

[0085]Dispersible powders and granules suitable for preparation of an
aqueous suspension by the addition of water generally contain the active
ingredient together with a dispersing or wetting agent, suspending agent
and one or more preservatives. Suitable dispersing or wetting agents and
suspending agents are exemplified by those already mentioned above.
Additional excipients such as sweetening, flavouring and colouring
agents, may also be present.

[0086]The pharmaceutical compositions of the invention may also be in the
form of oil-in-water emulsions. The oily phase may be a vegetable oil,
such as olive oil or arachis oil, or a mineral oil, such as for example
liquid paraffin or a mixture of any of these. Suitable emulsifying agents
may be, for example, naturally-occurring gums such as gum acacia or gum
tragacanth, naturally-occurring phosphatides such as soya bean, lecithin,
an esters or partial esters derived from fatty acids and hexitol
anhydrides (for example sorbitan monooleate) and condensation products of
the said partial esters with ethylene oxide such as polyoxyethylene
sorbitan monooleate. The emulsions may also contain sweetening,
flavouring and preservative agents.

[0087]Syrups and elixirs may be formulated with sweetening agents such as
glycerol, propylene glycol, sorbitol, aspartame or sucrose, and may also
contain a demulcent, preservative, flavouring and/or colouring agent.

[0088]The pharmaceutical compositions may also be in the form of a sterile
injectable aqueous or oily suspension, which may be formulated according
to known procedures using one or more of the appropriate dispersing or
wetting agents and suspending agents, which have been mentioned above. A
sterile injectable preparation may also be a sterile injectable solution
or suspension in a non-toxic parenterally-acceptable diluent or solvent,
for example a solution in 1,3-butanediol.

[0089]Compositions for administration by inhalation may be in the form of
a conventional pressurised aerosol arranged to dispense the active
ingredient either as an aerosol containing finely divided solid or liquid
droplets. Conventional aerosol propellants such as volatile fluorinated
hydrocarbons or hydrocarbons may be used and the aerosol device is
conveniently arranged to dispense a metered quantity of active
ingredient.

[0091]The amount of active ingredient that is combined with one or more
excipients to produce a single dosage form will necessarily vary
depending upon the host treated and the particular route of
administration. For example, a formulation intended for oral
administration to humans will generally contain, for example, from 0.5 mg
to 2 g of active agent compounded with an appropriate and convenient
amount of excipients which may vary from about 5 to about 98 percent by
weight of the total composition. Dosage unit forms will generally contain
about 1 mg to about 500 mg of an active ingredient. For further
information on Routes of Administration and Dosage Regimes the reader is
referred to Chapter 25.3 in Volume 5 of Comprehensive Medicinal Chemistry
(Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990.

[0092]According to a further aspect of the present invention there is
provided a compound of formula (I) or a pharmaceutically acceptable salt
thereof as defined hereinbefore for use in a method of treatment of the
human or animal body by therapy.

[0093]Reference herein to a compound of formula (I) should be understood
to refer equally to compounds of formula (I).

[0094]We have found that compounds of the present invention inhibit DGAT1
activity and are therefore of interest for their blood glucose-lowering
effects.

[0095]A further feature of the present invention is a compound of formula
(I) or a pharmaceutically-acceptable salt thereof for use as a
medicament.

[0096]Conveniently this is a compound of formula (I), or a
pharmaceutically-acceptable salt thereof, for use as a medicament for
producing an inhibition of DGAT1 activity in a warm-blooded animal such
as a human being.

[0097]Particularly this is a compound of formula (I), or a
pharmaceutically-acceptable salt thereof, for use as a medicament for
treating diabetes mellitus and/or obesity in a warm-blooded animal such
as a human being.

[0098]Thus according to a further aspect of the invention there is
provided the use of a compound of formula (I), or a
pharmaceutically-acceptable salt thereof in the manufacture of a
medicament for use in the production of an inhibition of DGAT1 activity
in a warm-blooded animal such as a human being.

[0099]Thus according to a further aspect of the invention there is
provided the use of a compound of formula (I), or a
pharmaceutically-acceptable salt thereof in the manufacture of a
medicament for use in the treatment of diabetes mellitus and/or obesity
in a warm-blooded animal such as a human being.

[0100]According to a further aspect of the invention there is provided a
pharmaceutical composition which comprises a compound of formula (I) as
defined hereinbefore or a pharmaceutically-acceptable salt thereof, in
association with a pharmaceutically-acceptable excipient or carrier for
use in producing an inhibition of DGAT1 activity in an warm-blooded
animal, such as a human being.

[0101]According to a further aspect of the invention there is provided a
pharmaceutical composition which comprises a compound of formula (I) as
defined hereinbefore or a pharmaceutically-acceptable salt thereof, in
association with a pharmaceutically-acceptable excipient or carrier for
use in the treatment of diabetes mellitus and/or obesity in an
warm-blooded animal, such as a human being.

[0102]According to a further feature of the invention there is provided a
method for producing an inhibition of DGAT1 activity in a warm-blooded
animal, such as a human being, in need of such treatment which comprises
administering to said animal an effective amount of a compound of formula
(I) or a pharmaceutically-acceptable salt thereof as defined
hereinbefore.

[0103]According to a further feature of the invention there is provided a
method of treating diabetes mellitus and/or obesity in a warm-blooded
animal, such as a human being, in need of such treatment which comprises
administering to said animal an effective amount of a compound of formula
(I) or a pharmaceutically-acceptable salt thereof as defined
hereinbefore.

[0104]As stated above the size of the dose required for the therapeutic or
prophylactic treatment of a particular disease state will necessarily be
varied depending on the host treated, the route of administration and the
severity of the illness being treated. Preferably a daily dose in the
range of 1-50 mg/kg is employed. However the daily dose will necessarily
be varied depending upon the host treated, the particular route of
administration, and the severity of the illness being treated.
Accordingly the optimum dosage may be determined by the practitioner who
is treating any particular patient.

[0105]As stated above compounds defined in the present invention are of
interest for their ability to inhibit the activity of DGAT1. A compound
of the invention may therefore be useful for the prevention, delay or
treatment of a range of disease states including diabetes mellitus, more
specifically type 2 diabetes mellitus (T2DM) and complications arising
there from (for example retinopathy, neuropathy and nephropathy),
impaired glucose tolerance (IGT), conditions of impaired fasting glucose,
metabolic acidosis, ketosis, dysmetabolic syndrome, arthritis,
osteoporosis, obesity and obesity related disorders, (which include
peripheral vascular disease, (including intermittent claudication),
cardiac failure and certain cardiac myopathies, myocardial ischaemia,
cerebral ischaemia and reperfusion, hyperlipidaemias, atherosclerosis,
infertility and polycystic ovary syndrome); the compounds of the
invention may also be useful for muscle weakness, diseases of the skin
such as acne, Alzheimer's disease, various immunomodulatory diseases
(such as psoriasis), HIV infection, inflammatory bowel syndrome and
inflammatory bowel disease such as Crohn's disease and ulcerative
colitis.

[0106]In particular, the compounds of the present invention are of
interest for the prevention, delay or treatment of diabetes mellitus
and/or obesity and/or obesity related disorders. In one aspect, the
compounds of the invention are used for prevention, delay or treatment of
diabetes mellitus. In another aspect, the compounds of the invention are
used for prevention, delay or treatment of obesity. In a further aspect,
the compounds of the invention are used for prevention, delay or
treatment of obesity related disorders.

[0107]The inhibition of DGAT1 activity described herein may be applied as
a sole therapy or in combination with one or more other substances and/or
treatments for the indication being treated. Such conjoint treatment may
be achieved by way of the simultaneous, sequential or separate
administration of the individual components of the treatment.
Simultaneous treatment may be in a single tablet or in separate tablets.
For example such conjoint treatment may be beneficial in the treatment of
metabolic syndrome [defined as abdominal obesity (as measured by waist
circumference against ethnic and gender specific cut-points) plus any two
of the following: hypertriglyceridemia (>150 mg/dl; 1.7 mmol/l); low
HDLc (<40 mg/dl or <1.03 mmol/l for men and <50 mg/dl or 1.29
mmol/l for women) or on treatment for low HDL (high density lipoprotein);
hypertension (SBP≧130 mmHg DBP≧85 mmHg) or on treatment for
hypertension; and hyperglycemia (fasting plasma glucose≧100 mg/dl
or 5.6 mmol/l or impaired glucose tolerance or pre-existing diabetes
mellitus)--International Diabetes Federation & input from IAS/NCEP].

[0108]Such conjoint treatments may include the following main categories:

[0116]In addition to their use in therapeutic medicine, compounds of
formula (I) and their pharmaceutically-acceptable salts are also useful
as pharmacological tools in the development and standardisation of in
vitro and in vivo test systems for the evaluation of the effects of
inhibitors of DGAT1 activity in laboratory animals such as cats, dogs,
rabbits, monkeys, rats and mice, as part of the search for new
therapeutic agents.

[0117]As indicated above, all of the compounds, and their corresponding
salts, are useful in inhibiting DGAT1. The ability of the compounds of
formula (I), and their corresponding acid addition salts, to inhibit
DGAT1 may be demonstrated employing the following enzyme assay:

[0119]DGAT1 activity was assayed by a modification of the method described
by Coleman (Methods in Enzymology 1992, 209, 98-102). Compound at 1-10
μM was incubated with 0.4 μg membrane protein, 5 mM MgCl2, and
100 μM 1,2 dioleoyl-sn-glycerol in a total assay volume of 200 μl
in plastic tubes. The reaction was started by adding 14C oleoyl
coenzyme A (30 μM final concentration) and incubated at room
temperature for 30 minutes. The reaction was stopped by adding 1.5 mL
2-propanol:heptane:water (80:20:2). Radioactive triolein product was
separated into the organic phase by adding 1 mL heptane and 0.5 mL 0.1 M
carbonate buffer pH 9.5. DGAT1 activity was quantified by counting
aliquots of the upper heptane layer by liquid scintillography.

[0121]The ability of the compounds of formula (I), and their corresponding
pharmaceutically-acceptable acid salts, to inhibit DGAT1 may further be
demonstrated employing the following whole cell assays 1) and 2):

1) Measurement of Triglyceride Synthesis in 3T3 Cells

[0122]Mouse adipocyte 3T3 cells were cultured to confluency in 6 well
plates in new born calf serum containing media. Differentiation of the
cells was induced by incubating in medium containing 10% foetal calf
serum, 1 μg/mL insulin, 0.25 μM dexamethasone and 0.5 mM
isobutylmethyl xanthine. After 48 h the cells were maintained in medium
containing 10% foetal calf serum and 1 μg/mL insulin for a further 4-6
days. For the experiment, the medium was changed to serum-free medium and
the cells pre-incubated with compound solubilised in DMSO (final
concentration 0.1%) for 30 minutes. De novo lipogenesis was measured by
the addition of 0.25 mM sodium acetate plus 1 μCi/mL 14C-sodium
acetate to each well for a further 2 h (J. Biol. Chem., 1976, 251,
6462-6464). The cells were washed in phosphate buffered saline and
solubilised in 1% sodium dodecyl sulfate. An aliquot was removed for
protein determination using a protein estimation kit (Perbio) based on
the method of Lowry (J. Biol. Chem., 1951, 193, 265-275). The lipids were
extracted into the organic phase using a heptane:propan-2-ol:water
(80:20:2) mixture followed by aliquots of water and heptane according to
the method of Coleman (Methods in Enzymology, 1992, 209, 98-104). The
organic phase was collected and the solvent evaporated under a stream of
nitrogen. The extracts solubilised in iso-hexane:acetic acid (99:1) and
lipids separated via normal phase high performance liquid chromatography
(HPLC) using a Lichrospher diol-5, 4×250 mm column and a gradient
solvent system of iso-hexane:acetic acid (99:1) and
iso-hexane:propan-2-ol:acetic acid (85:15:1), flow rate of 1 mL/minute
according to the method of Silversand and Haux (1997). Incorporation of
radiolabel into the triglyceride fraction was analysed using a Radiomatic
Flo-one Detector (Packard) connected to the HPLC machine.

2) Measurement of Triglyceride Synthesis in MCF7 Cells

[0123]Human mammary epithelial (MCF7) cells were cultured to confluency in
6 well plates in foetal calf serum containing media. For the experiment,
the medium was changed to serum-free medium and the cells pre-incubated
with compound solubilised in DMSO (final concentration 0.1%) for 30
minutes. De novo lipogenesis was measured by the addition of 50 μM
sodium acetate plus 3 μCi/mL 14C-sodium acetate to each well for
a further 3 h (J. Biol. Chem., 1976, 251, 6462-6464). The cells were
washed in phosphate buffered saline and solubilised in 1% sodium dodecyl
sulfate. An aliquot was removed for protein determination using a protein
estimation kit (Perbio) based on the method of Lowry (J. Biol. Chem.,
1951, 193, 265-275). The lipids were extracted into the organic phase
using a heptane:propan-2-ol:water (80:20:2) mixture followed by aliquots
of water and heptane according to the method of Coleman (Methods in
Enzymology, 1992, 209, 98-104). The organic phase was collected and the
solvent evaporated under a stream of nitrogen. The extracts solubilised
in iso-hexane:acetic acid (99:1) and lipids separated via normal phase
high performance liquid chromatography (HPLC) using a Lichrospher diol-5,
4×250 mm column and a gradient solvent system of iso-hexane:acetic
acid (99:1) and iso-hexane:propan-2-ol:acetic acid (85:15:1), flow rate
of 1 mL/minute according to the method of Silversand and Haux (J.
Chromat. B, 1997, 703, 7-14). Incorporation of radiolabel into the
triglyceride fraction was analysed using a Radiomatic Flo-one Detector
(Packard) connected to the HPLC machine.

[0124]In the above other pharmaceutical composition, process, method, use
and medicament manufacture features, the alternative and preferred
embodiments of the compounds of the invention described herein also
apply.

EXAMPLES

[0125]The invention will now be illustrated by the following Examples in
which, unless stated otherwise:

(i) temperatures are given in degrees Celsius (° C.); operations
were carried out at room or ambient temperature, that is, at a
temperature in the range of 18-25° C. and under an atmosphere of
an inert gas such as argon;(ii) organic solutions were dried over
anhydrous magnesium sulfate; evaporation of solvent was carried out using
a rotary evaporator under reduced pressure (600-4000 Pa; 4.5-30 mmHg)
with a bath temperature of up to 60° C.;(iii) chromatography means
flash chromatography on silica gel; where a Biotage cartridge is referred
to this means a cartridge containing KP-SIL® silica, 60 Å,
particle size 32-63 mM, supplied by Biotage, a division of Dyax Corp.,
1500 Avon Street Extended, Charlottesville, Va. 22902, USA;(iv) in
general, the course of reactions was followed by TLC and reaction times
are given for illustration only;(v) yields are given for illustration
only and are not necessarily those which can be obtained by diligent
process development; preparations were repeated if more material was
required;(vi) where given, NMR data (1H) is in the form of delta values
for major diagnostic protons, given in parts per million (ppm) relative
to tetramethylsilane (TMS), determined at 300 or 400 MHz (unless
otherwise stated) using perdeuterio dimethyl sulfoxide (DMSO-d6) as
solvent, unless otherwise stated; peak multiplicities are shown thus: s,
singlet; d, doublet; dd, doublet of doublets; dt, doublet of triplets;
dm, doublet of multiplets; t, triplet, q, quartet; m, multiplet; br,
broad;(vii) chemical symbols have their usual meanings; SI units and
symbols are used;(viii) solvent ratios are given in volume:volume (v/v)
terms;(ix) mass spectra (MS) (loop) were recorded on a Micromass Platform
LC equipped with HP 1100 detector; unless otherwise stated the mass ion
quoted is (MH.sup.+);(x) LCMS (liquid chromatography-mass spectrometry)
were recorded on a system comprising Waters 2790 LC equipped with a
Waters 996 Photodiode array detector and

[0126]Micromass ZMD MS, using a Phenomenex® Gemini 5u C18 110A
50×2 mm column and eluting with a flow rate of 1.1 ml/min with 5%
(Water/Acetonitrile (1:1)+1% formic acid) and a gradient increasing from
0-95% of acetonitrile over the first 4 minutes, the balance (95-0%) being
water and where HPLC Retention Times are reported these are in minutes in
this system unless otherwise stated; unless otherwise stated the mass ion
quoted is (MH.sup.+);